Nucleic acids, such as DNA and RNA as well as a number of nucleic acid analogues such as PNA, HNA, MNA, ANA, LNA, INA, CNA, CeNA, TNA, (2′-NH)-TNA, (3′-NH)-TNA, α-L-Ribo-LNA, α-L-Xylo-LNA, β-D-Xylo-LNA, α-D-Ribo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, α-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, β-D-Ribopyranosyl-NA, α-L-Lyxopyranosyl-NA, 2′-R1-RNA, 2′-OR1-RNA (R1 being any substituent), α-L-RNA, α-D-RNA, β-D-RNA and others are capable of specifically hybridising to complementary nucleic acid strands. This specific recognition may be utilised to detect the presence of defined nucleic acid sequences for example for diagnostic purposes. It is possible to detect small differences between nucleic acid sequences. It is also possible to determine the amount of mRNA present of a given gene, and thereby describe the expression level of that particular gene.
Many of the available assays rely on the detection of a specific nucleic acid by a complementary detectable probe. It is often required to separate unbound probe from bound probe in order to detect the specific nucleic acid, and this usually requires several time-consuming separation steps.
DNA diagnostics is one of the fastest growing research areas, and now with a more detailed map of the human genome map available day for day aiming for the full sequence in detail, the interest in the field is expected to expand even further. A map of 1.42 million single nucleotide polymorphisms (SNPs) has been described and it has been estimated that at least 60,000 SNPs fall within the human exons.
Interestingly, the genomic DNA sequence differs in average in about one out of 1000 nucleotides between two human beings. Accordingly, specific DNA sequences may be useful for determining the identity of an individual. Furthermore, mutations may be indicative of predisposition to clinical conditions. A classic example in genetic diseases is Sickle cell anemia, a genetic defect caused by a change of a single base in a single gene—the beta-globin gene (GAG is changed to GTG at Codon 6). The search for sequences that only differ in one or two nucleobases, creates the need for tools for detecting nucleic acid sequences with high performance, that are fast and simple to conduct, and are cost effective.
Another major field in biology is methylation of the genome. Methylation of “CpG” duplets in the genome is a key component of epigenetic information and has shown to play an important function in imprinting as well as in a range of clinical situations including mental disorders, cancer biology and viral infections. The commonly used technique for detecting the methylation status of a gene in a sensitive manner is treating the genomic DNA with sodium bisulphite, converting all unmethylated Cytosines “C” into deoxyribose Uracils “dUs” while methylated Cytosines “MeC” are unreactive, followed by an amplification reaction. The bisulphite treatment of the genomic DNA converts the DNA into an “A-T” rich, essentially three-nucleotide genome (with the exception of “MeC” present in methylated “CpG” duplets) and the difference of a methylated or unmethylated “CpG” duplet is translated into a sequence difference (see FIG. 5). The DNA can be analysed post amplification, by sequencing, restriction enzyme digestion or by the presence or absence of bands on a gel after gel electrophoreses when using Methylation Specific PCR, MSP. Alternatively the DNA can be analysed Real-time PCR using MSP and unspecific dye.
The “A-T” rich nature of the bisulphite treated DNA provides a special challenge to primers and probes in an amplification assay due to the relatively low affinity of “A-T” rich DNAs and the difficult nature of distinguishing between a single “C” and a “T” nucleotide.
Different techniques to detect mutations in nucleic acid sequences for example includes single-stranded conformational analysis, denaturing gradient gel electrophoresis, heteroduplex analysis, chemical mismatch cleavage and direct sequencing. A review of these techniques has been given by Grompe (M. Grompe (1993) Nature Genetics, 5:111-117). Among the variety of techniques also fluorescence based methods are available.
The invention of Real-Time Polymerase Chain Reaction (RT-PCR) has revolutionized the way of determining the status of genes like expression (Real-Time Reverse Transcriptase Polymerase Chain Reaction) in a fast and precise manner, but the technique is also used for differentiating between similar targets. Despite many advances in the technology there is still an unmet need for improvements of these techniques.
Some of the most widely used probes today are Molecular beacons, TaqMan® probes (TaqMan is a registered trademark of Roche Molecular Systems, Inc), Scorpion primers and dual hybridization FRET probes (recently reviewed by Arya et al. (2005) Rev. Mol. Diagn. 5:209-219). Each of these technologies has advantages and disadvantages in relation to each other in different applications. Common to all of these technologies are the difficulty of design, often requiring assistance from computer software or specialists and prolonged optimisation to work (examples of software are programs like Primer Express®. available from Applied Biosytems, United States, Beacon Designer available from Premier Biosoft. and Primer3 Copyright (c) 1996, 1997, 1998, 1999, 2000, 2001, 2004 Whitehead Institute for Biomedical Research. All rights reserved.
The TaqMan® assay uses a DNA based probe comprising a signalling pair consisting of a fluorophore and a quencher. The method preferably requires the fluorophore and the quencher to be positioned in opposite ends of the probe to achieve the best available signal-to-noise ratios. During amplification in the PCR reaction, the probe specifically hybridises to its target sequence that is being amplified. When reaching the TaqMan® probe, the Taq polymerase degrades the probe by using its exonuclease activity, whereby the fluorophore is free to move further away from the quencher eliminating the quenching of the signal. The fluorescence generated by the exonuclease activity is correlated to the amount of target sequence present. By knowing the efficiency of the PCR reaction the fluorescent signal can be used to calculate the starting amount of target sequence.
There have been reported some changes to the TaqMan® probes in order to optimise the specificity and sensitivity of the TaqMan® assay. These changes includes the introduction of Minor Groove Binders and nucleotide analogues such as Locked Nucleic Acid (see further description and references herein below) that was used to increase the affinity to the target sequence and to be able to use shorter probes. However in all of the reported TaqMan® assays, quenching of the fluorophore of the intact probe relies on random coil helix formation
The Molecular Beacon (“hairpin” or “stem-loop” probes) comprises two stem sequences, one in each end of a probe, which are able to form a duplex if there is no target sequence available for hybridisation complementary to the sequence separating (the loop sequence) said two stem sequences. The probe usually comprises a signalling pair consisting of a fluorophore and a quencher linked to opposite ends of the probe. When the stem is formed, the fluorophore and quencher are thus in close proximity and the signal is quenched. However if the loop sequence is hybridised to a target sequence, the two stem sequences and thus the fluorophore and quencher are separated, allowing a signal to be generated and read. Since there is no or very little background signal of the probe, it has been claimed that Molecular Beacons can be used for the detection of target sequences in homogenous assays and living cells.
The stem sequences of the Molecular Beacons are unrelated to the target sequence. Proper stem formation and stability is dependent upon the length and G:C content of the stem as well as the conditions of the buffer which it is dissolved in. As target sequences may also vary in G:C content, length and in how similar non-target sequences that might be present, the length and G:C content of the loop sequence will vary from probe to probe. It is therefore necessary to use optimized stem sequences that depends on the loop sequences. Furthermore the Molecular Beacons shall not only compete against reannealing of an amplicon, but also the intra-molecular annealing of the probe.
There have also been published some prior art regarding Stemless Beacons (Linear Beacons) including DNA and PNA based Stemless Beacons. The DNA Stemless beacons (Myanard, US 2005/0227247) are rendered impervious to digestion by 5′ to 3′ exonuclease activity and 3′ extension activity by a polymerase. The quenching of the fluorophore, placed in one end of the probe by the quencher placed in the other end of said probe, when the probe is unbound is controlled by random helix coil formation. Maynard teaches that the reporter molecule and quencher molecule are preferably separated by 18 or 20 bases to achieve optimal signal-to-noise ratio. The document and its prior art claims that said ratio is strongly dependent on length of the probe. Resistance to nuclease digestion is an inherent part of these stemless beacons, and the probes can't therefore be used in nuclease dependent assays like TaqMan® assays.
Gildea et al. (WO 1999/21881) describes PNA based Linear Beacons comprising a signalling pair with the two parts of said pair in each end of the probe. The difference between this invention and the invention of DNA based Linear Beacons are that the backbone monomer unit in Gildea's case is the backbone monomer unit of PNA and not DNA. They teach us that the reduced background of the probe is due to PNAs way of behaving and that there is a clear non-equivalency of structure and function between nucleic acid and PNA probes of similar length and labelling configurations. It is stated that probes described in the document should be unaffected of two of the following things: Probe length, Mg2+ concentration, ion strength of the buffer and spectral overlap between fluorophore and quencher, the first three things normally being observed in PNA and a few other DNA analogue based probes only. The document does not describe oligonucleotide analogues comprising hydrophobic molecules inserted into probes. Resistance to nuclease digestion is also part of the probes described by the document (inherent in PNA), and the probes can't therefore be used in nuclease dependent assays like TaqMan® assays.
In the prior art nucleotides and nucleotide analogues comprising signalling pairs have been described:
Kutyavin and co-workers conjugated a MGB molecule to the 3′-end of the probe and used it in a 5′-nuclease PCR assay (Kutyavin et al. (2000) Nucleic Acids Res. 28: 655-661), and the same laboratory also described a system, called Eclipse, where the MGB is attached to the 5′-end of the probe and does not require nuclease activity of the polymerase Afonina, I. et al. (1997) Nucleic Acids Res. 25: 2657-2660). In both of these systems the polymorphic base (point of potential mismatch) should be placed approximately 5 nucleotides from the attachment site of the MGB, to yield the optimum discrimination between the matched and the mismatched target, this is in contrast to normal DNA based probes that have previously been shown to give the highest difference in melting temperature and hence the highest specificity, if the polymorphic base is placed in the middle of the probe. The quenching of the fluorescence when the probe is unbound is limited to random coiling. The restricted design options can be a disadvantage as it is often necessary to have a high degree of flexibility in the probe design in order to avoid formation of unwanted secondary structures.
Letertre et al. 2003, showed that Locked Nucleic Acid, LNA, could be used in a 5′-nuclease PCR assay, with results comparable to the ones obtained with MGB TaqMan® probes (Letertre, C. et al., (2003)Mol. Cell. Probes 17: 307-311). It is however difficult to design optimal LNA containing probes based on two reasons: Firstly the LNA nucleotides are nuclease resistant, and the probe needs to be degraded during the 5′-nuclease assay and secondly LNA nucleotides have an extremely high self-affinity and hence if not carefully designed tend to form secondary structures. It has previously been shown that a DNA-LNA hybrid with only a few LNA nucleotides can form secondary structures that are so stable that the probe is unable to hybridise with complementary, single stranded DNA targets (Filichev, et al. (2004) Chem-biochem. 5:1673-1679).
Seitz, O. (2000) Angew. Chem. Int Ed. 39: 3249-3252) described Stemless Peptide Nucleic Acid (PNA) beacons. The PNA beacons tend to fold up into compact random coiled helixes when unbound to target nucleic acids, which is frequently undesirable. Even though that PNA based technologies offer some advantages over DNA based technologies in some aspects, they are more expensive than DNA based technologies and confers more challenge in synthesizing and finding optimal conditions than for DNA based assays. As PNAs are composed of a combination of a protein and an oligonucleotide, the behaviour of PNAs is not always readily predictable.
WO 2004/033726, describes the measurement of fluorescence in a sample being amplified in the presence of a fluorescent system consisting of a probe with a fluorophore attached and free floating quencher molecules that are double stranded intercalators.
WO 1999/21881, US 2002/0064772 and EP 1484337, describe the linear beacons comprising PNA and related molecules that are insensitive to ionic strength, magnesium concentration, spectral overlap between fluorophore and quencher and length of probe. Probes comprising hydrophobic molecules are not included or discussed in these patents.
US 2005/0233360, describes a probe with a fluorophore and a quencher in close proximity in the same end of the probe. Upon hybridisation to a complementary nucleic acid will either the fluorophore or the quencher interact with the formed duplex (by intercalation or minor groove binding) and the fluorophore will be able to fluoresce without being quenched.
WO 01/73118, describes a probe comprising a fluorophore and optionally a quencher. The fluorophore will behave differently depending on if a mismatch is present in the target nucleic acid or not and is in this way able to distinguish one from the other. The probe can also be used for melting point analysis. The probe usually has to be blocked in the 3′-end to avoid extension.
US 2005/0227247, describes the use of a probe with a fluorophore in one end and a quencher in the other end, where the probe is protected from 5′ to 3′ exonuclease degradation by one or more inserts of phosphorothioates in the 3′ end of the probe. Quenching of the fluorophore takes place due to random coiling.
US 2005/0227257, describes the use of a probe duplex, where one strand contains a fluorophore and the other strand contains a quencher. One of the strands is longer than the other and the quencher and fluorophore will be placed in close proximity if the two strands of the probe pair bind to each other. However in presence of a fully complementary target, the longer probe will preferentially bind to that, and the fluorophore will thus be separated from the quencher and hence allowed to fluoresce. Quenching of the probes is controlled by double helix formation of the two parts of the probe system.
US 2005/0064463, describes probes that optionally may be labelled with a fluorophore and a quencher as well as comprising a minor groove binder (MGB) at the end of the probe. The MGB increases the affinity for complementary DNA and shorter and more specific probes can therefore be used.
US 1999/5925517 and US 2000/6103476, describe the hairpin probes called “molecular beacons”. The probes comprise a fluorophore and a quencher one in each ends of an oligonucleotide or oligonucleotide analogue. Said oligonucleotide or oligonucleotide analogue is divided into the sequences, sequence 1 and 3 make up the stem in the hairpin structure, when the probe is not bound to any target nucleic acids and sequence 2 is complementary to the target nucleic acid. The background fluorescence is dependent on the double helix formation of sequence 1 and 3.
WO 03/052132, describes hairpin probes comprising Intercalating pseudonucleotides to increase affinity for target nucleic acids, increase nuclease stability, as fluorescent tags and/or decrease the self-affinity. The invention and description does not discuss the concept of “Stemless Beacons” according to the present invention. The document also describes the use of complementary probes for the detection of target nucleic acids, but contrary to the findings in the present invention it does not describe Stemless quenching of an unbound probe.
US 2000/6037130, describes triple labelled hairpin probes with a shorter wavelength harvester and a longer wavelength emitter and a quencher. The presence of the extra fluorophore compared to standard molecular beacons increases the fluorescent yield and hence the signal-to-noise ratio.
US 2002/6355421, describes methods, kits and composition of hairpin shaped PNA molecular beacons. The document does not describe the concept of Stemless beacons.
US 2005/0158720, describes Tripartite molecular beacons (TMBs), that comprise three oligonucleotide components one forming a hairpin structure and the two others complementary to the respective sides of the formed stem.
All patent and non-patent references cited in the application, are also hereby incorporated by reference in their entirety.